Modified proteins comprising controllable intervening protein sequences or their elements methods of producing same and methods for purification of a target protein comprised by a modified protein

A method of producing a target protein comprising:

• (a) producing a first modified protein wherein the amino-terminal portion of a controllable intervening protein sequence is fused to the carboxyl-terminus of the target protein;
• (b) producing a second modified protein comprising a portion of the controllable intervening protein sequence;
• and,
• (c) contacting the first and second modified proteins under a condition suitable for cleavage of the controllable intervening protein sequence in trans.

A method of producing a target protein comprising:

• (a) producing a first modified protein wherein the carboxyl-terminal portion of a controllable intervening protein sequence is fused to the amino terminus of the target protein;
• (b) producing a second modified protein comprising a portion of the controllable intervening protein sequence;
• and,
• (c) contacting the first and second modified proteins under a condition suitable for cleavage of the controllable intervening protein sequence in trans.

A method for purification of a target protein comprising:

• (a) forming a fusion protein comprising a controllable intervening protein sequence positioned between a target protein and a binding protein or portion thereof having affinity for a substrate;
• (b) contacting the fusion protein with a substrate to which the binding protein or portion thereof binds;
• (c) subjecting the substrate bound fusion protein to a condition suitable for the cleavage of the controllable intervening protein sequence or the cyclization of the target protein;
• and,
• (d) recovering the target protein.

A method for purification of a target protein comprising:

• (a) forming a fusion protein comprising a target protein fused to a controllable intervening protein sequence into which a binding protein or portion thereof is fused internally or terminally;
• (b) contacting the fusion protein with a substrate to which the binding protein or portion thereof binds;
• (c) subjecting the substrate bound fusion protein to a condition suitable for the cleavage of the controllable intervening protein sequence or the cyclization of the target protein;
• and,
• (d) recovering the target protein.

A method for purification of a target protein comprising:

• (a) forming a fusion protein comprising a controllable intervening protein sequence and a target protein;
• (b) contacting the fusion protein with a substrate to which the controllable intervening protein sequence binds;
• (c) subjecting the substrate bound fusion protein to a condition suitable for the cleavage of the controllable intervening protein sequence or for the cyclization of the target protein, permitting separation of the target protein from the controllable intervening protein sequence;
• and,
• (d) recovering the target protein.

A method for purification of a target protein comprising:

• (a) forming a fusion protein comprising a portion of a controllable intervening protein sequence inserted between a target protein and a binding protein or portion thereof having affinity for a substrate;
• (b) contacting the fusion protein with a substrate to which the binding protein or portion thereof binds;
• (c) contacting the substrate-bound fusion protein with a portion of the controllable intervening protein sequence;
• (d) subjecting the substrate-bound fusion protein and the portion of the controllable intervening protein sequence to a condition suitable for cleavage of the controllable intervening protein sequence in trans, thus permitting separation of the target protein from the controllable intervening protein sequence;
• and
• (e) recovering the target protein.

A method for purification of a target protein comprising:

• (a) forming a fusion protein comprising a target protein fused to a controllable intervening protein sequence into which a binding protein or portion thereof is fused internally or terminally;
• (b) contacting the fusion protein with a substrate to which the binding protein or portion thereof binds;
• (c) contacting the substrate-bound fusion protein with a portion of the controllable intervening protein sequence;
• (d) subjecting the substrate-bound fusion protein and the portion of the controllable intervening protein sequence to a condition suitable for cleavage of the controllable intervening protein sequence in trans, thus permitting separation of the target protein from the controllable intervening protein sequence;
• and
• (e) recovering the target protein.

A method for the purification of a target protein comprising:

• (a) forming a fusion protein comprising a portion of a controllable intervening protein sequence inserted between a target protein and a binding protein or portion thereof having affinity for a substrate;
• (b) contacting the fusion protein with a substrate to which the binding protein or portion thereof binds;
• (c) recovering the fusion protein of step (b);
• (d) contacting the recovered fusion protein of step (c) with a portion of the controllable intervening protein sequence;
• (e) subjecting the recovered fusion protein and the remaining portion of the controllable intervening protein sequence to a condition suitable for cleavage of the controllable intervening protein sequence in trans, thus permitting separation of the target protein from the binding protein or portion thereof;
• and
• (f) recovering the target protein.

A method for the purification of a target protein comprising:

• (a) forming a fusion protein comprising a target protein fused to a controllable intervening protein sequence into which a binding protein or portion thereof is fused internally or terminally;
• (b) contacting the fusion protein with a substrate to which the binding protein or portion thereof portion binds;
• (c) recovering the fusion protein of step (b);
• (d) contacting the recovered fusion protein of step (c) with a portion of the controllable intervening protein sequence;
• (e) subjecting the recovered fusion protein and a portion of the controllable intervening protein sequence to a condition suitable for cleavage of the controllable intervening protein sequence in trans, thus permitting separation of the target protein from the binding protein or portion thereof;
• and,
• (f) recovering the target protein.

A method for synthesizing a target protein comprising:

• (a) cloning or synthesizing a DNA encoding a first portion of the target polypeptide;
• (b) forming a genetic fusion of the 3'-terminus of the DNA encoding a first portion of the target polypeptide with the 5'-terminus of a second DNA encoding a controllable intervening protein sequence or element thereof to form a continuous open reading frame;
• (c) transforming a suitable host cell with the fused DNA of step (b) and culturing the transformed host cell under conditions suitable for the expression of the encoded fusion polypeptide;
• (d) isolating the fusion polypeptide from the host cell or its culture medium;
• (e) contacting the fusion polypeptide of step (d) with a thiol compound to induce cleavage of the fusion polypeptide and to produce a thiol ester intermediate at the carboxyl terminus of the target polypeptide;
• (f) contacting the thiol ester intermediate of step (e) with a second portion of the target polypeptide having an amino-terminal cysteine to form a peptide bond between the first and second portions of the target polypeptide;
• and
• (g) recovering the target polypeptide.

A method for labeling a target protein comprising:

• (a) cloning a DNA encoding a target polypeptide;
• (b) forming a genetic fusion of the 3'-terminus of the DNA encoding the target polypeptide with the 5'-terminus of a second DNA encoding a controllable intervening protein sequence or element thereof to form a continuous open reading frame;
• (c) transforming a suitable host cell with the fused DNA of step (b) and culturing the transformed host cell under conditions suitable for the expression of the encoded fusion polypeptide;
• (d) isolating the fusion polypeptide from the host cell or its culture medium;
• and,
• (e) contacting the fusion polypeptide of step (d) with a cysteine labeled at its sulfhydryl group or at its carboxyl group.

A method for binding and eluting a phage-displayed polypeptide from a target protein comprising:

• (a) forming a fusion protein comprising a target protein, a controllable intervening protein sequence or an amino-terminal or a carboxyl-terminal portion of a controllable intervening protein sequence, and a polypeptide capable of binding specifically to a solid support;
• (b) binding the fusion protein of step (a) to the solid support;
• (c) contacting a phage-displayed polypeptide with the support-bound fusion protein of step (b) permitting binding of the phage-displayed polypeptide with the fusion protein;
• (d) removing unbound phage-displayed polypeptide;
• and,
• (e) eluting the bound phage-displayed polypeptide by inducing cleavage of the controllable intervening protein sequence.